Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme.
Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
1989-10-01
|
| Series: | Journal of Lipid Research |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520382456 |
| _version_ | 1818383747035168768 |
|---|---|
| author | R T Fischer S H Stam P R Johnson S S Ko R L Magolda J L Gaylor J M Trzaskos |
| author_facet | R T Fischer S H Stam P R Johnson S S Ko R L Magolda J L Gaylor J M Trzaskos |
| author_sort | R T Fischer |
| collection | DOAJ |
| description | Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with unsaturation at either the delta 7 or delta 8 position in the sterol can be demethylated. The 14 alpha-methyl group of either lanostan-3 beta-ol or delta 6 lanostenol is not oxidized to the anticipated C-32 alcohol or aldehyde by the enzyme, nor are the corresponding 32-oxy-lanostanols demethylated when incubated with microsomal preparations. Despite the lack of metabolism, the saturated and delta 6 sterol analogues are effective competitive inhibitors of demethylase activity. Utilizing preferred substrates, comparison of the component reactions of the demethylation sequence shows that both the oxidative function and lyase function are sensitive to common inhibitors and that both activities require NADPH. These findings strongly support the premise that a P-450 isozyme does catalyze each phase of the lanosterol 14 alpha-methyl demethylation sequence. Collectively these results demonstrate the double-bond requirement for both components of the demethylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation. This participation may involve stabilizing a transition state intermediate or directing activated oxygen insertion as part of the P-450 monoxygenase mechanism. |
| first_indexed | 2024-12-14T03:11:16Z |
| format | Article |
| id | doaj.art-be121a8f8ce44d12911cb30e21189e16 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-14T03:11:16Z |
| publishDate | 1989-10-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-be121a8f8ce44d12911cb30e21189e162022-12-21T23:19:16ZengElsevierJournal of Lipid Research0022-22751989-10-01301016211632Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme.R T Fischer0S H Stam1P R Johnson2S S Ko3R L Magolda4J L Gaylor5J M Trzaskos6Medical Products Department, E.I. du Pont de Nemours and Co., Wilmington, DE 19880-0400.Medical Products Department, E.I. du Pont de Nemours and Co., Wilmington, DE 19880-0400.Medical Products Department, E.I. du Pont de Nemours and Co., Wilmington, DE 19880-0400.Medical Products Department, E.I. du Pont de Nemours and Co., Wilmington, DE 19880-0400.Medical Products Department, E.I. du Pont de Nemours and Co., Wilmington, DE 19880-0400.Medical Products Department, E.I. du Pont de Nemours and Co., Wilmington, DE 19880-0400.Medical Products Department, E.I. du Pont de Nemours and Co., Wilmington, DE 19880-0400.Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with unsaturation at either the delta 7 or delta 8 position in the sterol can be demethylated. The 14 alpha-methyl group of either lanostan-3 beta-ol or delta 6 lanostenol is not oxidized to the anticipated C-32 alcohol or aldehyde by the enzyme, nor are the corresponding 32-oxy-lanostanols demethylated when incubated with microsomal preparations. Despite the lack of metabolism, the saturated and delta 6 sterol analogues are effective competitive inhibitors of demethylase activity. Utilizing preferred substrates, comparison of the component reactions of the demethylation sequence shows that both the oxidative function and lyase function are sensitive to common inhibitors and that both activities require NADPH. These findings strongly support the premise that a P-450 isozyme does catalyze each phase of the lanosterol 14 alpha-methyl demethylation sequence. Collectively these results demonstrate the double-bond requirement for both components of the demethylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation. This participation may involve stabilizing a transition state intermediate or directing activated oxygen insertion as part of the P-450 monoxygenase mechanism.http://www.sciencedirect.com/science/article/pii/S0022227520382456 |
| spellingShingle | R T Fischer S H Stam P R Johnson S S Ko R L Magolda J L Gaylor J M Trzaskos Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme. Journal of Lipid Research |
| title | Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme. |
| title_full | Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme. |
| title_fullStr | Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme. |
| title_full_unstemmed | Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme. |
| title_short | Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme. |
| title_sort | mechanistic studies of lanosterol 14 alpha methyl demethylase substrate requirements for the component reactions catalyzed by a single cytochrome p 450 isozyme |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520382456 |
| work_keys_str_mv | AT rtfischer mechanisticstudiesoflanosterol14alphamethyldemethylasesubstraterequirementsforthecomponentreactionscatalyzedbyasinglecytochromep450isozyme AT shstam mechanisticstudiesoflanosterol14alphamethyldemethylasesubstraterequirementsforthecomponentreactionscatalyzedbyasinglecytochromep450isozyme AT prjohnson mechanisticstudiesoflanosterol14alphamethyldemethylasesubstraterequirementsforthecomponentreactionscatalyzedbyasinglecytochromep450isozyme AT ssko mechanisticstudiesoflanosterol14alphamethyldemethylasesubstraterequirementsforthecomponentreactionscatalyzedbyasinglecytochromep450isozyme AT rlmagolda mechanisticstudiesoflanosterol14alphamethyldemethylasesubstraterequirementsforthecomponentreactionscatalyzedbyasinglecytochromep450isozyme AT jlgaylor mechanisticstudiesoflanosterol14alphamethyldemethylasesubstraterequirementsforthecomponentreactionscatalyzedbyasinglecytochromep450isozyme AT jmtrzaskos mechanisticstudiesoflanosterol14alphamethyldemethylasesubstraterequirementsforthecomponentreactionscatalyzedbyasinglecytochromep450isozyme |